Histochemical analyses of living mouse liver under different hemodynamic conditions by "in vivo cryotechnique"

Abstract

Although the morphology and molecular distribution in animal liver tissues have been examined using conventional preparation methods, the findings are always affected by the technical artifacts caused by perfusion-fixation and tissue-resection. Using "in vivo cryotechnique" (IVCT), we have examined living mouse livers with histochemical, immunohistochemical and ultrastructural analyses. In samples prepared by IVCT, widely open sinusoids with many flowing erythrocytes were observed under normal blood circulation, and their collapse or blood congestion was seen in ischemic or heart-arrested mice. In contrast, the sinusoidal cavities were artificially dilated by perfusion-fixation, and collapsed by immersion-fixation and quick-freezing (QF) methods of resected tissues. The immunoreactivity of serum albumin and immunoglobulin G and intensity of periodic acid-Schiff-staining in hepatocytes were well preserved with the QF method and IVCT. Furthermore, following tissue resection, serum proteins were rapidly translocated into hepatocytes as demonstrated by immunoreactions on QF tissues frozen 1 or 5 min after resection. Translocation was not observed in IVCT samples, indicating that IVCT could be useful to examine cell membrane permeability of hepatocytes under different pathological conditions. Both dynamic morphology and immunodistribution of soluble components in living mouse livers, reflecting their physiological and pathological states, can be precisely examined by IVCT with higher time-resolution.